Amyotrophic lateral sclerosis (ALS) is a uniformly fatal neurodegenerative disease for which there is still no effective treatment. One explanation for this is that very few therapeutic targets - molecular events in the disease pathway whose inhibition confers benefit - have been identified, hindering rational translational approaches. One means of discovering novel targets is high-throughput in vitro screening of chemical libraries to identify compounds that can ameliorate disease-related phenotypes. Enzymes inhibited by such compounds constitute candidate therapeutic targets. However, to validate them for further development, it is necessary to demonstrate that their inhibition delays disease onset or progression in vivo. Among the cellular events that occur early in the ALS disease process is the physical die-back of motor axons from motor endplates, leading directly to muscle paralysis that spreads progressively throughout the body. Early muscle denervation is observed in mutant SOD1 rodent models as well as in patients with either sporadic or familial forms of the disease. Preventing axonal degeneration, or stimulating regrowth, would be predicted to delay disease onset or progression. However, since the underlying mechanisms remain unclear it has not been possible to test this therapeutic hypothesis directly. We recently screened a library of ~50,000 compounds to identify agents that enhance motor axon regeneration in an inhibitory context in vitro. The strongest hits were the statins, which enhanced axonal growth by up to 5- fold at concentrations 100-fold lower than with benchmarking compounds. Statin effects on axonal growth depend entirely on inhibition of their known target enzyme HMG-CoA reductase (HMGCR; 3-hydroxy-3-methyl- glutaryl-CoA reductase), which is the rate-limiting step for cholesterol synthesis and protein prenylation pathways. Our data identify HMGCR as a novel candidate therapeutic target in ALS. In this two-year project, we propose to validate HMGCR as a target in vivo using two potent ligands, cerivastatin and simvastatin, as probes. Our experiments will use compartmentalized motor neuron cultures to determine whether HMGCR inhibition needs to occur in the cell body or in the axon terminal. We will then establish protocol for statin administration in vivo that lead to significant HMGCR inhibition in motor neurons in the spinal cord. Finally, we will determine whether statin administration can delay muscle denervation in the mutant SOD1 mouse model of ALS. Overall, our experiments should allow us to determine whether HMGCR and the pathways downstream of it are valid therapeutic targets in ALS. They should also provide proof of principle for the idea that enhancing motor axon growth can delay functional denervation of muscle. Lastly, our data should stimulate further research into the specific processes modulated by statins in motor neurons, and thereby help identify more selective targets - such as protein prenylation - for future drug development.